Mechanisms regulating clathrin-mediated endocytosis

网格蛋白介导的内吞作用的调节机制

• 批准号: 8830980
• 负责人: Gaudenz Danuser
• 金额: $ 41.71万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8830980
• 关键词: Achievement; Adhesions; Affect; Algorithmic Software; Algorithms; Behavior; Biological Assay; Cardiovascular Diseases; Cell Surface Receptors; Cell membrane; Cell physiology; Cells; Clathrin; Clathrin-Coated Vesicles; Complex; Computer Simulation; Consensus; Data; Defect; Detection; Development; Diabetes Mellitus; Disease; Dynamin; Endocytosis; Engineering; Environment; Equilibrium; Event; Fluorescence Microscopy; Funding; Generations; Goals; Grant; Growth; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Image; Image Analysis; Individual; Kinetics; Label; Life; Link; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Measures; Mediating; Membrane; Methods; Microscopy; Modeling; Molecular; Neurologic; Nutrient; Pathology; Pathway interactions; Pattern; Physiology; Play; Population; Process; Proteins; Regulation; Relative (related person); Role; Signal Transduction; Slave; Small Interfering RNA; Staging; Surface; Testing; Transcription Factor AP-2 Alpha; Variant; Work; base; cell motility; cellular imaging; coated pit; feeding; genome editing; human disease; hypercholesterolemia; imaging modality; insight; interdisciplinary collaboration; interest; mathematical model; model building; molecular dynamics; nervous system disorder; novel therapeutics; particle; programs; protein function; quantitative imaging; receptor; sensor; uptake

DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis (CME) is the major pathway for internalization from, and remodeling of the plasma membrane (PM) in mammalian cells. Thus, CME plays a fundamental role in all aspects of cell physiology, including nutrient uptake, signal transduction, cell motility, adhesion, polarity and differentiation. Consequently, defects in CME impinge on many human diseases, including cancer, cardiovascular disease, diabetes, neurological defects and others. CME is a multistep process initiated by the assembly of clathrin and the AP2 adaptor complex to form nascent CCPs. Subsequent steps including CCP stabilization, maturation and their scission from the PM to release clathrin coated vesicles are orchestrated by a myriad of endocytic accessory proteins (EAPs). Under the auspices of this grant, we have built an interdisciplinary team of biochemists, cell and molecular biologists, microscopists, engineers and computational biologists whose long- term goal is to define the mechanisms that regulate CME. Over the past 8 years, we have developed accurate and highly sensitive particle detection and tracking software and algorithms to quantitatively measure multiple orthogonal parameters relating to the dynamics of clathrin-eGFP labeled CCPs imaged by live cell total internal reflection fluorescence microscopy. We identified 3 kinetically-distinc subpopulations of CCPs, two short-lived abortive populations and longer-lived productive CCPs, as well as factors that differentially affect CCP initiation, stabilization and maturation. These data led us to propose that CCP maturation is gated during the first ~30s after initiation by an "endocytic check-point" that is, in part, regulated by the GTPase dynamin. Early recruitment of AP2-interacting EAPs to CCPs is also required for efficient curvature generation and to regulate multiple stages of the maturation process. The overarching goal of this proposal is to test this checkpoint hypothesis by identifying the determinants (i.e. individual or sets of EAPs) that function as potential sensors or effectors of the checkpoint, as well as those required for efficient CCP stabilization and maturation. To accomplish these goals, we propose three Specific Aims: 1) To measure the effect of individual and pairwise knockdown of EAPs on stages of CME through quantitative multi-parametric analyses of CCP dynamics; 2) To develop a molecularly explicit, mathematical model of the multi-step CCP maturation program and calibrate model parameters against measured CCP lifetime distributions in siRNA-treated cells, and 3) To, directly test, under minimally perturbing conditions, the functional assignments of EAPs predicted from the studies in Aim 1 and 2, and define the functional hierarchy of EAPs in regulating CCP maturation and progression beyond the endocytic checkpoint. These studies will provide unprecedented insights into the function of individual EAPs and their role in regulating key early stages of CCP stabilization of maturation. While most studies have focused on readily detectable early (CCP initiation) or late (CCV budding) events, we focus here on the less well understood stages of CCP stabilization and maturation that are central to the regulation of CME.

描述（由申请人提供）：网格蛋白介导的内吞作用（CME）是哺乳动物细胞中质膜（PM）的内化和重塑的主要途径。因此，CME 在细胞生理学的各个方面发挥着重要作用，包括营养吸收、信号转导、细胞运动、粘附、极性和分化。因此，CME 缺陷会影响许多人类疾病，包括癌症、心血管疾病、糖尿病、神经系统缺陷等。 CME 是由网格蛋白和 AP2 接头复合物组装形成新生 CCP 引发的多步骤过程。随后的步骤，包括 CCP 稳定、成熟以及它们从 PM 上分离以释放网格蛋白包被的囊泡，是由无数内吞辅助蛋白 (EAP) 精心策划的。在这笔资助的支持下，我们建立了一个由生物化学家、细胞和分子生物学家、显微镜学家、工程师和计算生物学家组成的跨学科团队，其长期目标是定义调节 CME 的机制。在过去的 8 年里，我们开发了准确、高灵敏度的颗粒检测和跟踪软件和算法，以定量测量与活细胞全内反射荧光显微镜成像的网格蛋白-eGFP 标记的 CCP 动力学相关的多个正交参数。我们确定了 3 个动力学上不同的 CCP 亚群、两个短寿命的流产群体和寿命较长的生产性 CCP，以及对 CCP 启动、稳定和成熟产生不同影响的因素。这些数据使我们提出，CCP 的成熟是在启动后的前 30 秒内由“内吞检查点”控制的，该检查点在一定程度上受 GTP 酶动力的调节。为了有效地产生曲率并调节成熟过程的多个阶段，还需要尽早将与 AP2 相互作用的 EAP 招募到 CCP 中。该提案的总体目标是通过识别作为检查点的潜在传感器或效应器的决定因素（即个体或一组 EAP）以及有效 CCP 稳定和成熟所需的决定因素来测试该检查点假设。为了实现这些目标，我们提出了三个具体目标：1）通过 CCP 动态的定量多参数分析来测量 EAP 的个体和成对敲低对 CME 阶段的影响； 2) 开发多步骤 CCP 成熟程序的分子明确数学模型，并根据 siRNA 处理的细胞中测得的 CCP 寿命分布校准模型参数，以及 3) 在最小扰动条件下直接测试EAPs 根据目标 1 和 2 中的研究进行预测，并定义了 EAPs 在调节 CCP 成熟和超越内吞检查点的进展方面的功能层次。这些研究将为个体 EAP 的功能及其在调节 CCP 稳定成熟的关键早期阶段中的作用提供前所未有的见解。虽然大多数研究都集中在易于检测的早期（CCP 启动）或晚期（CCV 出芽）事件，但我们在这里关注的是不太为人所知的 CCP 稳定和成熟阶段，这些阶段是 CME 调节的核心。